Injection Of Lipopolysaccharide Research Articles

High sugar diet alters immune function and the gut microbiome in juvenile green iguanas (Iguana iguana).

The present work aimed to study whether a high sugar diet can alter immune responses and the gut microbiome in green iguanas. Thirty-six iguanas were split into four treatment groups using a 2x2 design. Iguanas either received a sugar supplemented diet or a control diet, and either received a lipopolysaccharide (LPS) injection or a phosphate buffer solution (PBS) injection. Iguanas were given their respective diet treatment through the entire study (∼3 months) and received a primary immune challenge one month and two months into the experiment. Blood samples and cloacal swabs were taken at various points in the experiment and used to measure changes in the immune system (bacterial killing ability, lysis and agglutination scores, LPS specific IgY concentrations), and alterations in the gut microbiome. We found that sugar diet reduces bacterial killing ability following an LPS challenge, and sugar and the immune challenge temporarily alters gut microbiome composition while reducing alpha diversity. While sugar did not directly reduce lysis and agglutination following the immune challenge, the change in these scores over a 24-hour period following an immune challenge was more drastic (it decreased) relative to the control diet group. Moreover, sugar increased constitutive agglutination outside of the immune challenges (i.e., pre-challenge levels). In this study, we provide evidence that a high sugar diet affects the immune system of green iguanas (in a disruptive manner) and alters the gut microbiome.

Boric Acid Alleviates Lipopolysaccharide-Induced Acute Lung Injury in Mice.

Acute lung injury (ALI) poses a significant medical challenge due to its widespread occurrence and high mortality rates. Despite extensive efforts, current clinical interventions for ALI have shown limited success. Inflammation plays a central role within ALI progress, and boric acid (BA) has demonstrated anti-inflammatory properties both in vitro and in vivo. However, its potential to mitigate lipopolysaccharide (LPS)-induced ALI remains an area awaiting exploration in research. To bridge this research gap, we created a mouse model of ALI induced by intraperitoneal LPS injection. We employed a comprehensive set of evaluation criteria, including H&E staining, wet/dry ratio measurement, malondialdehyde (MDA)/superoxide dismutase (SOD) the oxidative stress-related biomarkers, assessment of alveolar edema, hemorrhage, inflammatory cell infiltration, and examination of thickened alveolar septum to quantify lung injury. Additionally, we measured inflammatory cytokine levels using ELISA and assessed Nrf2 and HO-1 expressions through western blotting and quantitative real-time PCR (RT-PCR). ER stress-related markers (GRP78, CHOP) were analyzed through western blot analysis. Our findings revealed that prophylactic treatment with BA effectively attenuated LPS-induced ALI, as supported by improved pathological alterations, decreased total protein concentration in bronchoalveolar lavage fluid (BALF), and reduced pulmonary edema. Furthermore, BA exhibited anti-inflammatory properties by suppressing inflammatory cytokines within the lung tissue. BA ingestion caused upregulation in SOD and a decrease in MDA contents in lung tissue homogenates. BA downregulated the levels of GRP78 and CHOP compared to the LPS group. Remarkably, BA also upregulated transcription and protein expression of Nrf2 and HO-1 compared to the LPS group. In conclusion, our study highlights BA's potential as a novel promising prophylactic agent for LPS-induced ALI, offering avenue for improving clinical management of this condition.

#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.

Clostridium butyricum upregulates GPR109A/AMPK/PGC-1α and ameliorates acute pancreatitis-associated intestinal barrier injury in mice.

Acute pancreatitis frequently causes intestinal barrier damage, which aggravates pancreatitis. Although Clostridium butyricum exerts anti-inflammatory and protective effects on the intestinal barrier during acute pancreatitis, the underlying mechanism is unclear. The G protein-coupled receptors 109A (GPR109A) and adenosine monophosphate-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) signaling pathways can potentially influence the integrity of the intestinal barrier. Our study generated acute pancreatitis mouse models via intraperitoneal injection of cerulein and lipopolysaccharides. After intervention with Clostridium butyricum, the model mice showed reduced small intestinal and colonic intestinal barrier damage, dysbiosis amelioration, and increased GPR109A/AMPK/PGC-1α expression. In conclusion, Clostridium butyricum could improve pancreatic and intestinal inflammation and pancreatic injury, and relieve acute pancreatitis-induced intestinal barrier damage in the small intestine and colon, which may be associated with GPR109A/AMPK/PGC-1α.

Sestrin2 remedies neuroinflammatory response by inhibiting A1 astrocyte conversion via autophagy.

Most central nervous diseases are accompanied by astrocyte activation. Autophagy, an important pathway for cells to protect themselves and maintain homeostasis, is widely involved in regulation of astrocyte activation. Reactive astrocytes may play a protective or harmful role in different diseases due to different phenotypes of astrocytes. It is an urgent task to clarify the formation mechanisms of inflammatory astrocyte phenotype, A1 astrocytes. Sestrin2 is a highly conserved protein that can be induced under a variety of stress conditions as a potential protective role in oxidative damage process. However, whether Sestrin2 can affect autophagy and involve in A1 astrocyte conversion is still uncovered. In this study, we reported that Sestrin2 and autophagy were significantly induced in mouse hippocampus after multiple intraperitoneal injections of lipopolysaccharide, with the elevation of A1 astrocyte conversion and inflammatory mediators. Knockdown Sestrin2 in C8-D1A astrocytes promoted the levels of A1 astrocyte marker C3 mRNA and inflammatory factors, which was rescued by autophagy inducer rapamycin. Overexpression of Sestrin2 in C8-D1A astrocytes attenuated A1 astrocyte conversion and reduced inflammatory factor levels via abundant autophagy. Moreover, Sestrin2 overexpression improved mitochondrial structure and morphology. These results suggest that Sestrin2 can suppress neuroinflammation by inhibiting A1 astrocyte conversion via autophagy, which is a potential drug target for treating neuroinflammation.

Dietary L-carnitine supplementation modifies blood parameters of mid-lactating dairy cows during standardized lipopolysaccharide-induced inflammation.

L-carnitine, available as feed additive, is essential for the beta-oxidation of free fatty acids in the mitochondrial matrix. It provides energy to immune cells and may positively impact the functionality of leukocytes during the acute phase response, a situation of high energy demand. To test this hypothesis, German Holstein cows were assigned to a control group (CON, n = 26) and an L-carnitine supplemented group (CAR, n = 27, rumen-protected L-carnitine product: 125 g/cow/d, corresponded to total L-carnitine intake: 25 g/cow/d, supplied with concentrate) and received an intravenous bolus injection of lipopolysaccharides (LPS, 0.5 µg/kg body weight, E. coli) on day 111 postpartum as a model of standardized systemic inflammation. Blood samples were collected from day 1 ante injectionem until day 14 post injectionem (pi), with frequent sampling through an indwelling venous catheter from 0.5 h pi to 12 h pi. All parameters of the white blood cell count responded significantly to LPS, while only a few parameters were affected by L-carnitine supplementation. The mean eosinophil count, as well as the percentage of basophils were significantly higher in CAR than in CON over time, which may be due to an increased membrane stability. However, phagocytosis and production of reactive oxygen species by leukocytes remained unchanged following L-carnitine supplementation. In conclusion, although supplementation with 25 g L-carnitine per cow and day resulted in increased proportions of specific leukocyte populations, it had only minor effects on the functional parameters studied in mid-lactating dairy cows during LPS-induced inflammation, and there was no evidence of direct improvement of immune functionality.

Caffeic acid phenethyl ester restores mitochondrial homeostasis against peritoneal fibrosis induced by peritoneal dialysis through the AMPK/SIRT1 pathway

Increasing evidence suggests that peritoneal fibrosis induced by peritoneal dialysis (PD) is linked to oxidative stress. However, there are currently no effective interventions for peritoneal fibrosis. In the present study, we explored whether adding caffeic acid phenethyl ester (CAPE) to peritoneal dialysis fluid (PDF) improved peritoneal fibrosis caused by PD and explored the molecular mechanism. We established a peritoneal fibrosis model in Sprague–Dawley rats through intraperitoneal injection of PDF and lipopolysaccharide (LPS). Rats in the PD group showed increased peritoneal thickness, submesothelial collagen deposition, and the expression of TGFβ1 and α-SMA. Adding CAPE to PDF significantly inhibited PD-induced submesothelial thickening, reduced TGFβ1 and α-SMA expression, alleviated peritoneal fibrosis, and improved the peritoneal ultrafiltration function. In vitro, peritoneal mesothelial cells (PMCs) treated with PDF showed inhibition of the AMPK/SIRT1 pathway, mitochondrial membrane potential depolarization, overproduction of mitochondrial reactive oxygen species (ROS), decreased ATP synthesis, and induction of mesothelial-mesenchymal transition (MMT). CAPE activated the AMPK/SIRT1 pathway, thereby inhibiting mitochondrial membrane potential depolarization, reducing mitochondrial ROS generation, and maintaining ATP synthesis. However, the beneficial effects of CAPE were counteracted by an AMPK inhibitor and siSIRT1. Our results suggest that CAPE maintains mitochondrial homeostasis by upregulating the AMPK/SIRT1 pathway, which alleviates oxidative stress and MMT, thereby mitigating the damage to the peritoneal structure and function caused by PD. These findings suggest that adding CAPE to PDF may prevent and treat peritoneal fibrosis.

Effect on the splenocyte function of weaned piglets induced by continuous lipopolysaccharide injections

Abstract Introduction When piglets are exposed to pathogens for a long period, the immune system organs, among them the spleen, play a major role in combating the stress caused by those pathogens. In the present study, the effect on splenocyte function was investigated in a model of weaned piglets in which stress was induced by multiple low doses of lipopolysaccharide (LPS). Material and Methods Forty-eight 28-day-old piglets were divided into two groups: the LPS group and the control group. During the experimental period of thirteen days, the LPS group was intraperitoneally injected with LPS (100 μg/kg) once per day, and the control group was injected with the same volume of 0.9% sterile saline. On the 1st, 5th, 9th and 13th days, the piglets’ spleens were collected for isolating splenocytes. The proliferation ability of splenocytes was evaluated by the cell-counting-kit 8 method. Flow cytometry was used to detect cell cycle stage and apoptosis, and the nitric oxide level of cell supernatant was also tested. Results In the experimental group, the proliferation ability of splenocytes was enhanced, the proportion of cells in the G0/G1 phase was smaller, and cells were promoted to the S and G2/M phases. Meanwhile, apoptosis was suppressed and nitric oxide release upregulated. The results were significantly different between the LPS group and the control group on the 5th and 9th days. Conclusion The difference between the results of one group and those of the other suggest that after the 5th LPS injection, multiple low doses of LPS activated splenocytes and restored the number of splenocytes, which maintained and possibly enhanced the regulation of the immune function of the spleen.

Pineapple (Ananas comosus) Ameliorates Depressant-like Behaviors in Rats Induced by Lipopolysaccharide

Abstract Background: Lipopolysaccharide (LPS) injection can lead to depression and mood disorders by reducing tryptophan availability through increased activity of the enzyme indoleamine 2,3-dioxygenase. Tryptophan, sourced from fruits such as pineapple (Ananas cosmosus), is vital for the body, especially the serotonergic system. Aim: This study aims to assess how pineapple pulp (PP) counters LPS-induced depressive behaviors in male Wistar rats. Methods: The experiment involved force-feeding PP for 28 days before LPS injection (0.5 mg/kg intraperitoneal [i.p.]) in 30 male Wistar rats, divided into five groups. Groups I (normal) and II (LPS control) received 15 ml aquadest/kg/day. Test Groups III, IV, and V were given varied doses of PP; low dose (3.75 ml/kg/day), medium dose (7.5 ml/kg/day), and high dose (15 ml/kg/day). At the study's end, rats underwent the open field test (OFT) and the tail suspension test (TST). In addition, blood and brain samples were analyzed for neuroendocrine markers related to depression. Results: LPS triggers depressive symptoms, as evidenced by increased immobility time in the TST and altered behaviors indicative of anxiety in the OFT, especially in groups not receiving PP. This is followed by decreased serotonin levels in both plasma and brain when compared to groups given PP. In addition, higher corticosterone levels were observed in the LPS group than in the PP-treated group. Administering PP at 7.5 ml/kg/day for 28 days can alleviate the depressive effects induced by LPS injection. Conclusion: PP may have antidepressant properties, potentially by mitigating behaviors leading to depressive symptoms.

Central angiotensin-(1–7) attenuates hypoglycemia in sepsis-like conditions via reducing systemic and hepatic inflammation

Sepsis is understood as the result of initiating systemic inflammation derived from an inadequate host response against pathogens. In its acute phase, sepsis is marked by an exacerbated reaction to infection, tissue damage, organ failure, and metabolic dysfunction. Among these, hypoglycemia, characterized by disorders of the gluconeogenesis pathway, is related to one of the leading causes of mortality in septic patients. Recent research has investigated the involvement of sympathetic efferent neuroimmune pathways during systemic inflammation. These pathways can be stimulated by several centrally administered drugs, including Angiotensin-(1–7) (Ang-(1–7)). Therefore, the present study aims to evaluate the effects of central treatment with Ang-(1–7) on hypoglycemia during endotoxemia. For this, male Wistar Hannover rats underwent stereotaxic surgery for intracerebroventricular (i.c.v.) administration of Ang-(1–7) and cannulation of the jugular vein for lipopolysaccharide (LPS) injection. Our results demonstrate that LPS was capable of inducing hypoglycemia and that prior central treatment with Ang-(1–7) attenuated this effect. Our data also show that Ang-(1–7) reduced plasma concentrations of TNF-α, IL-1β, IL-6, and nitric oxide, in addition to the decrease and increase of hepatic IL-6 and IL-10 respectively, in animals subjected to systemic inflammation by LPS, resulting in the reduction of systemic and hepatic inflammation, thus attenuating the deleterious effects of LPS on phosphoenolpyruvate carboxykinase protein content. In summary, the data suggest that central treatment with Ang-(1–7) attenuates hypoglycemia induced by endotoxemia, probably through anti-inflammatory action, leading to reestablishing hepatic gluconeogenesis.

Neonatal immune stimulation results in sex-specific changes in ultrasonic vocalizations but does not affect seizure susceptibility in neonatal mice.

Neuroinflammation during the neonatal period has been linked to disorders such as autism and epilepsy. In this study, we investigated the early life behavioral consequences of a single injection of lipopolysaccharide (LPS) at postnatal day 10 (PD10) in mice. To assess deficits in communication, we performed the isolation-induced ultrasonic vocalizations (USVs) test at PD12. To determine if early life immune stimulus could alter seizure susceptibility, latency to flurothyl-induced generalized seizures was measured at 4 hours(hrs), 2days, or 5days after LPS injections. LPS had a sex-dependent effect on USV number. LPS-treated male mice presented significantly fewer USVs than LPS-treated female mice. However, the number of calls did not significantly differ between control and LPS for either sex. In male mice, we found that downward, short, and composite calls were significantly more prevalent in the LPS treatment group, while upward, chevron, and complex calls were less prevalent than in controls (p< 0.05). Female mice that received LPS presented a significantly higher proportion of short, frequency steps, two-syllable, and composite calls in their repertoire when compared with female control mice (p< 0.05). Seizure latency was not altered by early-life inflammation at any of the time points measured. Our findings suggest that early-life immune stimulation at PD10 disrupts vocal development but does not alter the susceptibility to flurothyl-induced seizures during the neonatal period. Additionally, the effect of inflammation in the disruption of vocalization is sex-dependent.

Programmed cell death factor 4-mediated hippocampal synaptic plasticity is involved in early life stress and susceptibility to depression

Early life stress (ELS) increases the risk of depression later in life. Programmed cell death factor 4 (PDCD4), an apoptosis-related molecule, extensively participates in tumorigenesis and inflammatory diseases. However, its involvement in a person’s susceptibility to ELS-related depression is unknown. To examine the effects and underlying mechanisms of PDCD4 on ELS vulnerability, we used a “two-hit” stress mouse model: an intraperitoneal injection of lipopolysaccharide (LPS) into neonatal mice was performed on postnatal days 7–9 (P7-P9) and inescapable foot shock (IFS) administration in adolescent was used as a later-life challenge. Our study shows that compared with mice that were only exposed to the LPS or IFS, the “two-hit” stress mice developed more severe depression/anxiety-like behaviors and social disability. We detected the levels of PDCD4 in the hippocampus of adolescent mice and found that they were significantly increased in “two-hit” stress mice. The results of immunohistochemical staining and Sholl analysis showed that the number of microglia in the hippocampus of “two-hit” stress mice significantly increased, with morphological changes, shortened branches, and decreased numbers. However, knocking down PDCD4 can prevent the number and morphological changes of microglia induced by ELS. In addition, we confirmed through the Golgi staining and immunohistochemical staining results that knocking down PDCD4 can ameliorate ELS-induced synaptic plasticity damage. Mechanically, the knockdown of PDCD4 exerts neuroprotective effects, possibly via the mediation of BDNF/AKT/CREB signaling. Combined, these results suggest that PDCD4 may play an important role in the ELS-induced susceptibility to depression and, thus, may become a therapeutic target for depressive disorders.

Exposure to normobaric hypoxia shapes the acute inflammatory response in human whole blood cells in vivo.

Cells of the immune defence, especially leukocytes, often have to perform their function in tissue areas that are characterized by oxygen deficiency, so-called hypoxia. Physiological hypoxia significantly affects leukocyte function and controls the innate and adaptive immune response mainly through transcriptional gene regulation via the hypoxia-inducible factors (HIFs). Multiple pathogens including components of bacteria, such as lipopolysaccharides (LPS) trigger the activation of leukocytes. HIF pathway activation enables immune cells to adapt to both hypoxic environments in physiological and inflammatory settings and modulates immune cell responses through metabolism changes and crosstalk with other immune-relevant signalling pathways. To study the mutual influence of both processes in vivo, we used a human endotoxemia model, challenging participants with an intravenous LPS injection post or prior to a 4-h stay in a hypoxic chamber with normobaric hypoxia of 10.5% oxygen. We analysed changes in gene expression in whole blood cells and determined inflammatory markers to unveil the crosstalk between both processes. Our investigations showed differentially altered gene expression patterns of HIF and target genes upon in vivo treatment with LPS and hypoxia. Further, we found evidence for effects of hypoxic priming upon inflammation in combination with immunomodulatory effects in whole blood cells in vivo. Our work elucidates the complex interplay of hypoxic and inflammatory HIF regulation in human immune cells and offers new perspectives for further clinical research.

237 Disrupted insulin signaling due to an acute immune response in swine

Abstract Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Altered insulin signaling can occur due to metabolic state, pathogen exposure, and autoimmune disorders. Lipopolysaccharide (LPS), an endotoxin secreted produced by gram-negative bacteria, can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. Crossbred nursery pigs [n = 58, initial body weight (BW) = 12.1 kg ± 0.23 kg] were randomly assigned into two experimental subsets, where 10 animals were designated for repeated blood sampling and 48 animals underwent timed euthanasia for tissue collection. For repeated blood sampling, jugular catheters were placed under anesthesia and the animals were given 48 h to recover before being randomly assigned to treatments. These treatments were an intraperitoneal injection of either 15µg/kg BW LPS or an equal volume of 0.9% Saline (n = 5 pigs/treatment). Blood was collected for 48 h followed by euthanasia. The other 48 pigs were randomly assigned to the same treatments, and then randomly assigned to euthanasia timepoints of 0, 3, 6, 12, 24 or 48 h post injection for tissue collection. Among the catheterized pigs, 2-h post LPS challenge there was a decrease in serum insulin concentration (P &amp;lt; 0.05) and mild hyperglycemia (P &amp;lt; 0.05) post challenge. At 12- and 24-h post LPS challenge there was a decrease in insulin (P &amp;lt; 0.05) and a trend hyperglycemia was also apparent at 24 h (P = 0.063). In liver tissue, insulin related genes exhibited alterations post LPS injection. Phosphoinositide 3-kinase (pi3k) and insulin receptor substrate 1 (irs1) expression were decreased at 3 and 6 h (p&amp;lt; 0.05) and protein kinase b (akt) expression were decreased at 3 h (P &amp;lt; 0.05) post challenge. The expression of insulin inhibitor growth factor receptor bound protein 10 (grb10) was decreased at 6-, 12-, and 24-h after challenge (P &amp;lt; 0.05). Fructose-1-6-bisphosphatase (f16bp) expression was reduced at 3-, 6-, and 12-h (P &amp;lt; 0.05) in the liver while phosphoenolpyruvate carboxykinase (pepck) expression was decreased at 3- and 6-h (P &amp;lt; 0.01) post challenge. Other metabolic genes were decreased at 3-, 6-, and 12-h post LPS challenge, including fatty acid synthase (fas; P &amp;lt; 0.05), carbohydrate response element binding protein (chrebp; P &amp;lt; 0.05), and acetyl CoA carboxylase (acc; P &amp;lt; 0.05). Protein abundance of Akt 3 h after LPS injection was decreased in the liver (P &amp;lt; 0.05). In skeletal muscle 6 h post LPS injection, insulin receptor (insr), insulin-like growth factor receptor (igfr) and fas were all increased (P &amp;lt; 0.05) These results indicate disrupted insulin signaling at various levels after an acute immune response.

180 Dietary milk fat globule membrane alters intestinal microbiota of neonatal piglet following lipopolysaccharide challenge and marker of neuroinflammation

Abstract Early life stress exposure can increase morbidity and reduce animal wellbeing throughout life. Dysregulated gut function has been associated with neurological changes, and neurological changes affect gut homeostasis. Maternal milk is the gold-standard for yielding developmental benefits to offspring. Identification of milk components has led to an understanding of the bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal gut health and neurodevelopment. However, mechanisms of actions are still being defined. The objective is to understand how dietary MFGM modulates the gut microbiota, blood-brain barrier tight junctions, and brain inflammatory markers following lipopolysaccharide (LPS) challenge. Piglets (n = 24, one-d-old) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) in a 2 × 2 factorial design (n = 6 animals•diet-1•challenge-1.). Piglets were fed for 21 d and 6 animals/diet received saline or LPS injections 4 h before euthanasia. Colonic microbiota was characterized through 16s rRNA gene sequencing, while hypothalamus samples were collected to evaluate brain barrier function and inflammation-related genes using RT-qPCR. Microbiota data were processed using QIIME2 with DADA2 plugin and analyzed for alpha-diversity, beta-diversity, and differential abundance in R software. Gene transcription data were analyzed by a 2 × 2 experimental factorial design using the PROC MIXED procedure of SAS. Principal coordinates analysis indicated that MFGM-fed groups exhibited an observable separation from CON-fed groups in terms of microbial community structures (P &amp;lt; 0.05). Firmicutes_A, Bacteroidota, and Firmicutes_D were the predominant phyla across all treatments, with average proportions to be 52.21 %, 24.36 %, and 18.46 %, respectively. At genus level, MFGM + Saline group down-regulated Corynebacterium by 88.00 % (q &amp;lt; 0.05) and Unclassified Muribaculaceae by 73.33 % (0.050.05). However, there was an MFGM × LPS interaction (P &amp;lt; 0.05) observed for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, in which MFGM attenuated the LPS-induced increase in mRNA relative abundance (3.62 and 2.39 ± 0.38). In conclusion, dietary MFGM altered neonatal piglet intestinal microbiota and an important signaling molecule of the neuroinflammatory response following LPS challenge.

Pomegranate polyphenol punicalin ameliorates lipopolysaccharide-induced memory impairment, behavioral disorders, oxidative stress, and neuroinflammation via inhibition of TLR4-NF-кB pathway.

Neuroinflammation may play an important role in the development of Alzheimer's disease (AD). Previous studies have reported that lipopolysaccharide (LPS)-induced neuroinflammation causes memory impairments and behavioral disorders. We investigated the potential preventive effects of punicalin (PUN), a polyphenolic component of pomegranate, on LPS-induced memory deficiency and anxiety- and depression-like behaviors, along with the underlying mechanisms. LPS-treated cultured microglial BV2 cells and BV2 cell/Neuro-2a (N2a) cell coculture system were investigated for anti-neuroinflammatory effects of PUN in vitro. The in vivo experiments involved mice administered a 4-week course of oral gavage with 1500 mg/kg/d PUN before intraperitoneal LPS (250 mg/kg daily 7 times) injections. The in vitro results demonstrated that PUN inhibited the LPS-induced inflammatory cytokine (IL-18, IL-1β, TNF-ɑ, and IL-6) production in BV2 cells and protected N2a cells from synaptic damage mediated by BV2 microglia-induced neuroinflammation. In in vivo studies, it was observed that PUN improved memory impairment and anxiety- and depression-like behaviors caused by LPS and reduced the expression of inflammatory proteins such as iNOS, COX-2, IL-1β, IL-2, IL-6, and TNF-α. Furthermore, PUN inhibited the LPS-induced production of MDA; increased the activities of CAT, SOD, and GSH-Px, and inhibited LPS-induced Aβ1-42 generation through down-regulation of APP and BACE1 expression. Moreover, PUN also suppressed the expression of TLR4, IRAK4, TRAF6, IKK-β, NF-κB, p65, and HMGB1 in LPS-treated mouse brain and cultured microglial BV-2 cells. These results suggest that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti-amylogenic mechanisms through inhibition of TLR4-NF-kB activation.

Differential Responses of Spinal Microglia to Low-Dose LPS Delivered in the Daily Rest vs Active Phase

Virtually all lung and neural disorders are associated with systemic and/or neuro-inflammation. Neuro-inflammation impairs important forms of neuroplasticity associated with cognitive and spinal motor learning ( e.g., respiratory motor plasticity). Microglia are resident CNS innate immune cells that regulate diverse processes including neuroinflammation and multiple forms of neuroplasticity via neuron-microglia interactions. Brain microglia exhibit profound diurnal rhythms in their response to pro-inflammatory stimuli, such as toll-like receptor 4 (TLR4) activation. Although hippocampal microglia develop a pro-inflammatory phenotype in response to TLR4 activation during the daily rest phase, little is known concerning spinal microglial responses to inflammatory stimuli across the daily rest/active cycle. Thus, we tested the hypothesis that ventral spinal microglia near the phrenic motor nucleus differentially respond to low-dose systemic lipopolysaccharide (LPS) delivered in the rest (12PM) vs active (12AM) phase. Specifically, we investigated microglial responses to systemic inflammation induced by low-dose LPS (0.1mg/kg, i.p.) in 2 populations of adult Sprague-Dawley rats (both 3-6 months old): 1) SD wildtype and 2) SD rats with humanized neuro-neutral apolipoprotein 3 (h APOE3) knock-in. All rats received intrapleural Cholera toxin B (CtB) injections 2 weeks prior to harvest to identify phrenic motor neurons. Twenty-four hours post-LPS or vehicle (0.9% saline), rats were perfused with 1X PBS and fixed with 4% PFA. Spinal tissues containing the phrenic motor nucleus (cervical 3-6) were harvested and dehydrated in 30% sucrose for immunohistochemistry. Activated microglia near phrenic motor neurons were visualized with an Iba-1 antibody and quantified and phenotyped based on morphology. Since no differences in microglia were found between wildtype and h APOE3 rats, data were combined into a single group for analyses. In vehicle-treated rats, robust Iba-1 staining near phrenic motor neurons was observed in the active, but not rest phase. In contrast, Iba-1 staining in LPS treated rats was elevated only when LPS was administered in the rest (not active) phase. After rest-phase LPS injections, microglial cell bodies were larger and had more processes compared to rest-phase controls. In the active phase, spinal microglia were similar between vehicle and LPS injected rats. Thus, spinal microglia exhibit a profound diurnal rhythm in (morphological) responses to systemic inflammation ( i.e. LPS). Further studies are needed to identify specific molecules driving time-of-day differences in spinal microglial responses to pro-inflammatory stimuli. Nevertheless, these findings increase our understanding of how diurnal rhythms impact cellular responses to physiological challenges, such as inflammation. Further, our results yield clues concerning diurnal variations in phrenic motor plasticity, since reciprocal phrenic motor neuron/microglial communication is an important regulator of phrenic motor plasticity. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electroacupuncture pre-treatment exerts a protective effect on LPS-induced cardiomyopathy in mice through the delivery of miR-381 via exosomes

ObjectiveThis study aims to investigate the cardiac protective effects and molecular mechanisms of electroacupuncture (EA) pre-treatment in lipopolysaccharide (LPS)-Induced Cardiomyopathy. Methods and resultsPre-treatment with EA was performed 30 min before intraperitoneal injection of LPS. Cardiac function changes in mice of the EA + LPS group were observed using electrocardiography, echocardiography, and enzyme linked immunosorbent assay (ELISA) and compared with the LPS group. The results demonstrated that EA pre-treatment significantly improved the survival rate of septic mice, alleviated the severity of endotoxemia, and exhibited notable cardiac protective effects. These effects were characterized by a reduction in ST-segment elevation on electrocardiography, an increase in ejection fraction (EF) and fraction shortening (FS) on echocardiography and a decrease in the expression of serum cardiac troponin I (cTn-I) levels. Serum exosomes obtained after EA pre-treatment were extracted and administered to septic mice, revealing significant cardiac protective effects of EA-derived exosomes. Furthermore, the antagonism of circulating exosomes in mice markedly suppressed the cardiac protective effects conferred by EA pre-treatment. Analysis of serum exosomes using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed a significant upregulation of miR-381 expression after EA pre-treatment. Inhibition or overexpression of miR-381 through serotype 9 adeno-associated virus (AAV9)-mediated gene delivery demonstrated that overexpression of miR-381 exerted a cardiac protective effect, while inhibition of miR-381 significantly attenuated the cardiac protective effects conferred by EA pre-treatment. ConclusionsOur research findings have revealed a novel endogenous cardiac protection mechanism, wherein circulating exosomes derived from EA pre-treatment mitigate LPS-induced cardiac dysfunction via miR-381.

Heparin-Binding Protein Promotes Acute Lung Injury in Sepsis Mice by Blocking the Aryl Hydrocarbon Receptor Signaling Pathway.

This study aimed to explore the therapeutic effect and potential mechanism of heparin-binding protein (HBP) reduction on sepsis-related acute lung injury. We utilized a murine model of sepsis-induced by intraperitoneal injection of lipopolysaccharides (LPS) in C57BL/6J mice divided into four groups: Control, LPS, Anti-HBP, and ceftriaxone (CEF). Following sepsis induction, Anti-HBP or CEF treatments were administered, and survival rates were monitored for 48 h. We then used reverse-transcription quantitative PCR to analyze the expression levels of HBP in lung tissues, immunohistochemistry for protein localization, and Western blotting for protein quantification. Pulmonary inflammation was assessed using enzyme-linked immunosorbent assays of proinflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β, IL-6, and interferon-γ). The activation state of the aryl hydrocarbon receptor (AhR) signaling pathway was determined via Western blotting, evaluating both cytoplasmic and nuclear localization of AhR and the expression of cytochrome P450 1A1 protein by its target gene. Anti-HBP specifically reduced HBP levels. The survival rate of mice in the Anti-HBP and CEF groups was much higher than that in the LPS group. The severity of lung injury and pulmonary inflammatory response in the Anti-HBP and CEF groups was significantly lower than that in the LPS group. AhR signaling pathway activation was observed in the Anti-HBP and CEF groups. Additionally, there was no significant difference in the above indices between the Anti-HBP and CEF groups. HBP downregulation in lung tissues significantly improved LPS-induced lung injury and the pulmonary inflammatory response, thereby prolonging the survival of sepsis mice, suggesting activation of the AhR signaling pathway. Moreover, the effect of lowering the HBP level was equivalent to that of the classical antibiotic CEF. Not applicable.

The role of ZC3H12D-regulated TLR4-NF-κB pathway in LPS-induced pro-inflammatory microglial activation

Lipopolysaccharide (LPS) is an important neurotoxin that can cause inflammatory activation of microglia. ZC3H12D is a novel immunomodulator, which plays a remarkable role in neurological pathologies. It has not been characterized whether ZC3H12D is involved in the regulation of microglial activation. The aim of this study was to investigate the role of ZC3H12D in LPS-induced pro-inflammatory microglial activation and its potential mechanism. To elucidate this, we established animal models of inflammatory injury by intraperitoneal injection of LPS (10 mg/kg). The results of the open-field test showed that LPS caused impaired motor function in mice. Meanwhile, LPS caused pro-inflammatory activation of microglia in the mice cerebral cortex and inhibited the expression of ZC3H12D. We also constructed in vitro inflammatory injury models by treating BV-2 microglia with LPS (0.5 μg/mL). The results showed that down-regulated ZC3H12D expression was associated with LPS-induced pro-inflammatory microglial activation, and further intervention of ZC3H12D expression could inhibited LPS-induced pro-inflammatory activation of microglia. In addition, LPS activated the TLR4-NF-κB signaling pathway, and this process can also be reversed by promoting ZC3H12D expression. At the same time, the addition of resveratrol, a nutrient previously proven to inhibit pro-inflammatory microglial activation, can also reverse this process by increasing the expression of ZC3H12D. Summarized, our data elucidated that ZC3H12D in LPS-induced pro-inflammatory activation of brain microglia via restraining the TLR4-NF-κB pathway. This study may provide a valuable clue for potential therapeutic targets for neuroinflammation-related injuries.